Nucleolaron1511 and Nucleolaron1510 Facilitate Effective Intracellular Delivery of Azami Green Protein in HeLa Cells

The intracellular delivery of large macromolecules remains a significant challenge in both therapeutic and research contexts. This study investigates two novel chimeric cell-penetrating peptides, Nucleolaron1511 (Nuc1511) and Nucleolaron1510 (Nuc1510), with the aim of evaluating their capacities to deliver the fluorescent protein Azami Green (AG) into HeLa cells via genetic fusion. Cellular uptake of TAMRA-labeled Nuc1510 was assessed by fluorescence microscopy at multiple time points. Delivery efficiency of AG–Nuc1511 and AG–Nuc1510 fusion proteins was evaluated by fluorescence microscopy and flow cytometry, and cytotoxicity was assessed across a range of concentrations using the MTT assay. TAMRA–Nuc1510 successfully penetrated HeLa cells within one hour of treatment, with signal detected in both cytoplasmic and nuclear compartments. Flow cytometry quantification revealed that fusion with Nuc1511 increased intracellular AG fluorescence by over 80-fold relative to unconjugated AG, while Nuc1510 fusion yielded approximately a 9-fold increase. Neither peptide exhibited significant cytotoxicity at concentrations used in the delivery assays. These findings establish Nuc1511 and Nuc1510 as effective CPPs capable of facilitating intracellular protein delivery via genetic fusion, with favorable safety profiles, representing promising candidates for protein-based intracellular delivery in research and therapeutic applications.